Fig. 1: XIST attenuates X chromosome transcription in naive hES cells.
From: XIST dampens X chromosome activity in a SPEN-dependent manner during early human development
a, Scheme of XIST depletion approach (CRISPR–Cas9 or CRISRPi) in primed H9 hES cells. b, Correlation heatmap generated using epiblast markers specific to each blastocyst stage from human preimplantation and postimplantation embryo single-cell RNA (scRNA)-seq data20,25. Numbers correspond to the embryonic days (E5 to E12). c, Percentage of biallelically expressed SNPs from X chromosome and chromosome 7 in naive (NaiveCult, n = 3; PXGL, n = 6) and primed (n = 3) H9 hES cells obtained from RNA-seq data. Box plots represent the median (center), first and third quartiles (hinges) and ±1.5 interquartile range (IQR; whiskers). d, Representative RNA FISH images for XIST and two X-linked genes (HUWE1 and POLA1) in naive (PXGL) and primed H9 hES cells. Percentages of cells displaying the representative pattern are indicated. Scale bar = 10 µm. e, Representative RNA FISH images for XIST in naive H9 hES cells with a stable or Dox-inducible XIST repression. Percentages of cells displaying the representative pattern are indicated. Scale bar = 10 µm. f, Quantification of RNA FISH data showing the percentage of cells with XIST signal in XIST WT, XIST KO and three XIST CRISPRi naive H9 clones (C1–C3) treated or not with Dox. g, RT–qPCR analysis of XIST expression in naive XIST CRISPRi clones with or without Dox treatment (left) and in XIST WT and KO naive and primed H9 hES cells (right). Values are normalized to β-actin. h, X:A ratio from RNA-seq data of XIST WT, KO, UT CRISPRi and Dox-treated (Dox) CRISPRi naive H9 cells (n = 3). Data are presented as the mean values ± s.d. of replicates. i, pXi allelic ratio from RNA-seq data of primed (WT) and naive (XIST WT, KO, UT CRISPRi and Dox-treated CRISPRi) H9 hES cells (n = 3). Data are presented as the mean values ± s.d. of replicates. j, X:A ratio from fastGRO-seq data of XIST UT and Dox-treated CRISPRi naive H9 cells (n = 3). Data are presented as the mean values ± s.d. of replicates. k, pXi allelic ratio from fastGRO-seq data of XIST UT and Dox-treated CRISPRi naive H9 cells (n = 3). Data are presented as the mean values ± s.d. of replicates. l, Ratio of the median coverage of reads on the TSS and gene body from fastGRO-seq data from XIST UT (n = 2) and Dox-treated (n = 3) CRISPRi naive H9 hES cells. Unless otherwise specified, P values were calculated by a two-sided unpaired t-test. Levels of significance: not significant (NS; P ≥ 0.05), *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
